For the purpose of the detection of specific nucleic acids (target nucleic acids), such as genetic diagnosis, identification of pathogenic bacteria, and the detection of single nucleotide polymorphisms, the hybridization between probe nucleic acid and target nucleic acid is employed. In recent years, DNA chips and DNA microarrays in which multiple probe nucleic acids are immobilized on a substrate have been put to practical use to detect target nucleic acids.
In the manufacturing of DNA chips and DNA microarrays, DNA must be arrayed in a form of multiple spots and immobilized on a substrate. For example, one method that is employed to immobilize DNA is to bind a thiol to single-stranded DNA and immobilize the thiolated single-stranded DNA on a metal substrate. The immobilized DNA is then subjected to the action with target DNA of a specimen and the presence or absence of hybrids is detected. For example, hybridization can be detected by detecting the fluorescence from each spot of immobilized DNA that have hybridized with target DNA.
Spotting-type DNA microarrays are prepared by placing liquid droplets containing probe DNA on a substrate and drying them. Thus, although they have the advantage of being inexpensive to produce, there is no guarantee that the DNA that is immobilized on the substrate will be uniform. That is, variations in size and shape of the DNA detection spots result in a drawback of spotting-type DNA microarrays. This drawback arises from, for example, the entire surface of a substrate being treated to immobilize DNA (PLL treatment) or the substrate surface being flat.
Further, in spotting-type DNA microarrays, the presence of solid-phase-forming agents adhering around the DNA detection spots is problematic in that it causes the target DNA to nonspecifically adsorb to the substrate, increasing noise and lowering the S/N ratio.
Further, in fluorescence measurements, an operation referred to as gridding is conducted to identify fluorescent components. In gridding, the number of rows and columns of spots on the array, the spot spacing, and the size of the spot diameter are inputted, and the spots are enclosed in circles (see “DNA Microarray Practice Manual that Will Necessarily Yield Data, Basic Principle, From Chip preparation to Bioinformatics”, 1st ed., Yodosha, Dec. 1, 2002, p. 19-21, 35, 106-108). However, when the stamp shape and position are inconstant, the gridding operation requires an extended time during fluorescence analysis and accurate analysis becomes difficult. Further, in gridding, spots cannot be accurately enclosed when the spot positions have shifted. Thus, software is imparted with an automatic position-correcting function. However, all operations are not automated; it is necessary to manually set the spot starting point and visually confirm and correct the grids of all the spots. This operation is extremely complicated. When the DNA spots amount to several thousands, the operation consumes time, thereby analysis speed.
Additionally, hybridization of sample target DNA to probe DNA immobilized on a substrate commonly requires well over ten hours. A large quantity of sample target DNA is also required. Thus, a large amount of time, expense, and effort are required for hybridization and the preparation of a large quantity of sample. In particular, an extremely large amount of target sample is required when analyzing low-expression genes.
Accordingly, it is one object of the present invention to provide a substrate having biomolecule immobilization regions of prescribed shape on a biomolecule microarray, and means by which the interaction of biomolecules, particularly the hybridization of nucleic acids, is rapidly conducted, the interaction of trace quantities of sample is promoted, and the interaction is detected and analyzed rapidly and with high sensitivity.
It is a further object of the present invention to provide a method and device of promoting interaction, by which interaction between biomolecules capable of interaction each other is promoted and efficiently formed.
A still further object of the present invention is to provide means for automatically conducting the gridding operation to permit automation of the collection of fluorescence data from a biomolecule microarray and automation of the digital analysis thereof.
More specifically, the present invention has for its objects to provide a substrate for biomolecule microarray permitting the detection of interaction between biomolecules with high sensitivity and permitting automated gridding, and to provide a biomolecule microarray in which biomolecules are immobilized on such a substrate.
A still further object of the present invention is to provide a method of detecting interaction between biomolecules permitting automated gridding.